Vascular Endothelial Growth Factor Promote Hematopoietic Differentiation From Murine Embryonic Stem Cells

Objective: Embryonic stem(ES) cells were extensively studied during the recent decade. The line of ES cells was first isolated from the inner cells mass of murine blastocysts. These cells are capable of preserving totipotency, undifferentiated state and normal diploid karyotype during long-term in vitro cultivation. ES cells have a potential to differentiate into all kinds of cells from ectoderm, mesoderm, and endoderm. After the introduction of an intact blastocyst in the cavity, the ES cells may take part in the formation of all tissues and organs of chimeric descendants, including the germ cells. The increased interest in ES cells is due to the possibilities in studies of the basic mechanisms underlying cell commitment and differentiation, pluripotency and the effects of endogenous and exogenous factors that determine the type of in vivo and in vitro differentiation. The most significant is the perspective of the use of ES cells for gene and cell therapy. Strictly conditions are need for in vitro culture of ES cells in order to maintain ES cells in a proliferative and undifferentiated state. In this article, we cultured mouse ES cells on the primary mouse embryonicfibroblasts(PMEF) or in culture medium supplemented with recombinant murine leukemia inhibitory factor(LIF). The research provide a solid foundation for the next work and the application of ES cells.Methods: Mouse ES cell line D3 was used in this study. The primary mouse embryonic fibroblasts(PMEF) was isolated and cultured for feeder layer cells from mouse fetus of gestational 13 to 14 day. ES cells(D3 line) were cultured in following culture systems: on the mitomycin C-treated PMEF feeder layer cells or LIF in culture medium consisting of Dulbecco's modified Eagle medium supplemented with 20% fetal calf serum , 1% nonessential amino acids, 0.1 mmol/L β -mercaptoethanol and 2 mmol/L glutamine. The ES cells growth state and clone formation in these two culture systems were observed morphologically by reverse microscope.Results: ES cells could grow, proliferate, and form colonies during in vitro culture in the presence of PMEF or LIF. When mitomycin C-treated PMEF was used as the feeder cells for co-culture with ES-cells, the ES cells could form nested colonies with clear edge and smooth surface as well as close arrangement within the colony. ES cells developed varied cell colonies when cultured in the medium contain LIF. The time formed cell colonies was longer than that cultured on PMEF. No sign of differentiation was seen in these two culture systems.Conclusion: Each of the two culture systems could be used to promote the growth and proliferation of ES cells, maintain their undifferentiation and pluripotentiality. It will benefit to the application of ES cells in next study.Key words: Part 2:Vascular endothelial growth factor promote CD34~+ cells generation from murine embryonic stem cellsObjective: Embryonic stem(ES) cells are totipotent cells from the inner cellmass of a blastocyst. In vivo, they could contribute to all the tissues of an animal, including germ cells, when transplanted back into a developing blastocyst. In vitro, their differentiation provides a model system to study the development of various tissues and cells. In particular, ES cells can differentiate into hematopoietic stem/progenitor cells, are another source for hematopoietic stem cells transplantation. In studies of hematopoiesis, investigators have used ES cells to derive various hematopoietic lineages in vitro either by suspension culture, formation of embryoid bodies or coculture with stromal cell lines. The vascular endothelial growth factor(VEGF) receptor Flk-1 is known to play a key role in the regulation of embryonic vascular and hematopoietic development. The aim of this study is to investigate the role of VEGF in promoting CD34+ cells generation from mouse ES cells in vitro.Methods: 1 n The culture of ES cells: A murine ES cell line, ES-D3, was maintain as undifferentiated cells by coculture with mitomycin C-treated PMEF feeder layer cells in culture medium consisting of Dulbecco's modified Eagle medium supplemented with 20% fetal calf serum , 1% nonessential amino acids, 0.1 mmol/L P -mercaptoethanol and 2 mmol/L glutamine. 2 ^ The formation of embryoid body(EB): ES cell colonies growth on PMEF were dissociated with 0.25% trypsin-EDTA , and single-cell suspension was made. The cells were seeded onto a feeder-free, 24-well plate at a concentration of 1 X105 cells /ml, and incubated at 37 °C in a humidified flushed with 5%CO2 in air atmosphere. After 4 days, the EBs were observed by reverse microscopy. 3, Experimental groups: EB cells were dissociated into single-cell suspension by 0.25% trypsin-EDTA treatment and mild pipetting, then were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF. The cells were incubated at 37°C in a humidified incubator containing 5%CO2 in air atmosphere for 1 week. Six groups, i.e. VEGF 5 u g/L, VEGF 10 u g/L, VEGF 20 u g/L, VEGF 5 u g/L+SCF 100 u g/L, VEGF 10 u g/L+SCF 100 u g/L and VEGF 20 u g/L+SCF 100 u g/L, were designed. The group of spontaneous differentiation without cytokine(s) used as control. 4>CD34mRNA expression: Cells were collected from the culture plate. Total RNA was extracted, RNA concentration of the samples quantified, and complementary DNA(cDNA) synthesized. CD34mRNA expression of each group was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The target sequence was approximately 398bp in length. Products were analyzed on 2% agarose gel and visualized with ethidium bromide staining. 5> Flow cytometry analysis: Collecting cells, making single-cell suspension, and were stained with fluorescein isothiocyanate(FITC)-conjugated rat anti-mouse CD34, then were assessed by flow cytometry. Ten thousand events were collected and analysed with CellQuestPlot software. 6 -. Hematopoietic colony assay: Hematotopoietic colonies were demonstrated by growing these cells in semisolid culture medium consisting of 0.9% methylcellulose, 30% fetal calf serum, 1%BSA, 10"4M P -mercaptoethanol, 2 mmol/L L-glutamine, 50 u g/ml rSCF, 20 ng/ml rGM-CSF, 20 ng/ml rIL-3, 20 ng/ml rIL-6, 20 ng/ml rG-CSF and 3 units/ml rEpo. Cells were aliquoted in triplicate samples at 2X105cell per plate, and incubated at 37°C in a humidified incubator containing 5 % CO2 in air atmosphere. After 2 weeks the plates were scored for total colony-forming units in culture(CFU-C) by reverse microscopy. 7^ Terminally differentiated cells detection: Collecting cell colonies cultured in semisolid methylcellulose culture medium, making cell smear, and were detected by using CD lib and Ter-119 immunocytochemistry detection kit. The positive cells which its membrane dyed with yellow-brown was observed.Results: 1 n Embryoid body(EB) formation: ES cells could grow and maintain their undifferentiated state by coculture with mitomycin C-treated PMEF. The cultivation of ES cells without a feeder layer for 4 days leads to the formation of embryoid bodyes. 2> CD34mRNA expression: CD34mRNA expression of each group was detected by RT-PCR. The results showed that undifferentiated ES cells and cells from the group of spontaneous differentiation without cytokine(s) did not express CD34mRNA. CD34mRNA could be detected in cells collected from EB and each experimental group. The relatively high level expression was emergent in those cellsfrom the group of VEGF 10 n g/L + SCF and VEGF 20 u g/L + SCF. 3 , CD34+ cells detection: The percent of CD34+ cells in each group were measured by flow cytometry. Results showed that VEGF combined with SCF can significantly promote CD34+ cells generation. The percent of CD34+ cells in group of VEGF 20 u g/L+ SCF and VEGF 10 u g/L+SCF was significantly higher than other groups(P <0. 05 or 0. 01). On the other hand, CD34+ cells could hardly be detected in the group of spontaneous differentiation. 4> Hematopoietic colony assay: After 2 weeks culture in semisolid methylcellulose medium, CFU-C could grow in group of EB and each experimental group, while otherwise in group of spontaneous differentiation. The numbers of CFC were significantly increased when VEGF combined with SCF be used. Compared with other groups, the numbers of CFC were highest in the group of VEGF 20ug/L + SCF and VEGF lOug/L + SCF (P <0. 05 or 0.01). 5, Terminally differentiated cells detection: The differentiated cells were detected by using CDllb and Ter-119 immunocytochemistry detection kit. The positive cells which its membrane dyed with yellow-brown can be observed.Conclusion: VEGF could promote the differentiation of murine ES cells into CD34+ cells in vitro. When cultured on semisolid medium with hematopoietic growth factors, those cells induced from ES cells could form hematopoietic colony and differentiate into terminal cells expressed surface antigen CDllb and Terll9. As a single factor, VEGF gave the stimulatory effect on the expression of CD34mRNA, the generation of CD34+ cells and CFU-C. The effects of VEGF+SCF were the most potent in the experimental groups according to the expression of CD34mRNA, the percent of CD34+cells and the numbers of hematopoietic colonies. The most highest inducing efficacy were achieved when VEGF 20 u g/L or VEGF 10 w g/L combined with SCF were used. The in vitro differentiation of ES cells provided an opportunity to better understand hematopoiesis and could lead to a novel source of cells for transfusion and transplantation therapies.Key words: Embryonic stem cells ; Vascular endothelial growth factor; Embryoid body; CD34~+ cellsPart 3: Study on the expression of genes associated withhematopoietic differentiation in murine embryonic stemcells induced by vascular endothelial growth factorObjective: Under appropriate culture conditions, ES cells undergo differentiation in vitro to form hematopoietic precursors. The hematopoietic character of these precursors can be supported further by the demonstration that they express genes associated with early hematopoietic differentiation. In an effort to characterize further the hematopoietic (stem/progenitor) cells derived from murine ES cell differentiation induced by vascular endothelial growth factor(VEGF), we initiated experiments to analyze the expression patterns of hematopoietic transcription factor GATA-2 and genes associated with early hematopoietic differentiation(c-kit and 3 -HI) during the differentiation process.Methods: 1> The culture of ES cells: A murine ES cell line, ES-D3, was maintain as undifferentiated cells by coculture with mitomycin C-treated PMEF feeder layer cells in culture medium consisting of Dulbecco's modified Eagle medium supplemented with 20% fetal calf serum , 1% nonessential amino acids, 0.1 mmol/L 3 -mercaptoethanol and 2 mmol/L glutamine. 2 -. The formation of embryoid body(EB): ES cell colonies growth on PMEF were dissociated with 0.25% trypsin-EDTA , and single-cell suspension was made. The cells were seeded onto a feeder-free, 24-well plate at a concentration of 1 X 105 cells /ml , and incubated at 37 °C in a humidified flushed with 5%CO2 in air atmosphere. After 4 days, the EBs were observed by reverse microscopy. 3^ Experimental groups: EB cells were dissociated into single-cell suspension by 0.25% trypsin-EDTA treatment and mild pipetting, then were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF. The cells were incubated at 37°C in a humidified incubator containing 5 % CO2 in air atmosphere for 1 week. Six groups, i.e. VEGF 5ug/L, VEGF lOug/L, VEGF 20ug/L, VEGF 5ug/L+SCF lOOug/L,VEGF 10 u g/L+SCF 10011 g/L and VEGF 20 u g/L+SCF 100 u g/L, were designed. The group of spontaneous differentiation without cytokine(s) was a control. 4> RT-PCR analysis: Cells were collected from the culture plate. Total RNA was extracted, RNA concentration of the samples quantified, and complementary DNA(cDNA) synthesized. GATA-2, c-kit and P -HImRNA expression of each group was identified by reverse transcriptase-polymerase chain reaction. Products were analyzed on 2% agarose gel and visualized with ethidium bromide staining. The relative coefficient of mRNA expression was also be analysed.Results: 1 ? Embryoid body(EB) formation: ES cells could grow and maintain their undifferentiated state by coculture with mitomycin C-treated PMEF. The cultivation of ES cells without a feeder layer for 4 days could be developed into embryoid bodies(EB). 2> Expression of GATA-2 mRNA: Expression of GATA-2 mRNA could be detected in every group of experiment after 1 week culture. It was expressed at very high levels in group of VEGF 20 u g/L and VEGE+SCF. The relative coefficient of GATA-2 mRNA expression in these groups was significantly higher than that in other groups(P<0.01). 3 > Expression of c-kit and ￡ -HlmRNA: Undifferentiated ES cells did not express c-kit and P -HI mRNA. Expression of c-kit mRNA could be detected in EB cells. The high levels were detected in group of VEGF 10 u g/L+SCF and VEGF 20 u g/L+SCF. There was significant differences compared with the other groups(P<0.05). Expression of & -HI mRNA was negative and weakly positive in group of VEGF 10 u g/L and VEGF 5 u g/L. Emergence of 3 -HI mRNA expression was evident when the cells cultured with VEGF 20ng/ml and VEGF+SCF. The most high expression was detected in group of VEGF lOng/ml+SCF. There was significant difference compared with other groups(/> <0.01 or 0.05). Expression of GATA-2, c-kit and P -HI mRNA was undetectable in group of spontaneous differentiation.Conclusion: Hematopoietic differentiation from ES cells can be induced by VEGF. The hematopoietic character of these cells derived from ES cells is supported by the demonstration that they express genes associated with hematopoietic...